Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can proliferate indefinitely in the undifferentiated state, and differentiate into many types of cells in human tissues, including the heart (Non-patent literatures 1-7). Therefore, hPSCs are potentially useful in cell-baaed therapies for heart disease. Efficient production of functional cardiac cells from hPSCs is required for cell-based therapy. The most common methods currently used are suspension culture of embryoid bodies with cytokines, such as DKK1, bFGF, Activin A, and EMP4, or adhesion co-culture with mouse END2 (visceral endoderm-like cells) (Non-patent literatures 13, and 15-17) However, these methods have low efficiencies (10-50% cardiomyocytes produced) (Non-patent literature 15), xeno-contamination is unavoidable with the use of animal cells or fetal bovine serum (FBS) (Non-patent literature 18), and use of recombinant cytokines is not cost-effective for large-scale production. Furthermore, a previous study showed that optimal cytokine concentrations for cardiac induction differ among individual hPSC lines (Non-patent literature 19), indicating the need for optimization procedures. A universal cardiac differentiation method that is independent of hPSC lines was recently reported, but it requires FBS and growth factors, such as bFGF and BMP4, for efficient differentiation (Non-patent literature 20).
Small molecules have great potential as substitutes for recombinant cytokines and unknown factors in FBS (Non-patent literature 21), and they are suitable for making defined media for large-scale culture. To date, small molecules have been used to activate or inhibit signaling pathways, such as WNT or TGF-β signaling (Non-patent literatures 22 and 23), or to regulate the expression of genes in place of transcription factors (Non-patent literatures 24 and 25). A number of small molecules have been examined or screened for promotion of differentiation: a BMP signaling inhibitor (Dorsomorphin), a p38MAPK signaling inhibitor (SB203580), a WNT signaling activator (BIO), and WNT signaling inhibitors (XAV939, IWR-1, IWP-1, and IWP-3) were reported to promote cardiac differentiation (Non-patent literatures 26-32). However, these treatments resulted in only 10-60% differentiation to cardiomyocytes, even under serum-containing conditions (Non-patent literatures 26-32). A small molecule that produces more efficient differentiation is needed for large-scale clinical applications.